Novel acyl CoA:cholesterol acyltransferase (ACAT-2)

ABSTRACT

Nucleic acid compositions encoding novel ACAT proteins, as well as the novel ACAT-2 proteins, (ACAT-2) are provided. Also provided are methods of producing the subject nucleic acid and protein compositions. The subject polypeptide and nucleic acid compositions find use in a variety of applications, including diagnostic and therapeutic agent screening applications, as well as in treatment therapies for disease conditions associated with ACAT-2 activity, e.g., in the treatment of gall stones.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part application of U.S.patent application Ser. No. 09/328,857, filed Jun. 8, 1999; whichapplication, pursuant to 35 U.S.C. § 119 (e), claims priority to thefiling date of the U.S. Provisional Patent Application Serial No.60/090,354 filed Jun. 23, 1998, the disclosure of which is hereinincorporated by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under Grant Nos.R01 52069 and H157170 awarded by the National Institute of Health. TheGovernment has certain rights in this invention.

INTRODUCTION

[0003] 1. Field of the Invention

[0004] The field of the invention is enzymes, particularlyacyltransferases.

[0005] 2. Background of the Invention

[0006] The ability to synthesize sterol esters is fundamental to mosteukaryotic cells. Sterol esterification is thought to participate in themaintenance of cell membrane sterols at levels optimal for normal cellfunction. In mammalian cells, cholesterol esterification is catalyzed bythe enzyme acyl CoA:cholesterol acyltransferase (E.C. 2.3.1.26, ACAT).

[0007] ACAT activity has been implicated in a number of physiologicprocesses. In the small intestine, ACAT has been proposed to play a rolein cholesterol absorption by maintaining a free cholesterol diffusiongradient across the enterocyte surface through the intracellularformation of cholesterol esters. Cholesterol ester formation by ACAT hasalso been hypothesized to be important for the assembly and secretion ofapolipoprotein B-containing lipoproteins in the intestine and the liver.In the adrenal glands and other steroidogenic tissues, ACAT synthesizescholesterol esters that accumulate in cytosolic droplets where they canserve as cholesterol substrate stores for steroidogenesis. Inmacrophages, ACAT generates intracellular cholesterol esters that arestored as cytosolic lipid droplets, a characteristic feature ofmacrophage foam cells in atherosclerotic lesions.

[0008] Recent evidence has suggested that more than one ACAT exists inmammals. A human ACAT cDNA was first identified from a macrophage cDNAlibrary. The disruption of the mouse homolog of this ACAT gene (Acact)yielded viable, ACAT-deficient (Acact^(−/−)) mice that werecharacterized by tissue-specific reductions in cholesterol esters.Cholesterol ester stores were markedly reduced in adrenal cortices andcultured peritoneal macrophages; however, substantial levels of ACATactivity were present in Acact^(−/−) livers, and intestinal cholesterolabsorption was normal, indicating that another ACAT enzyme was active inthese tissues. Studies examining the tissue distribution of Acact mRNAexpression also supported the hypothesis that more than one ACAT exists,as did previous biochemical and ACAT inhibitor studies showingdifferences between liver and aorta/macrophage ACAT activities. Theabove results indicate that a second ACAT enzyme contributes tocholesterol esterification activity in the liver and small intestine.

[0009] As such, there is much interest in the identification, isolationand characterization of this putative second ACAT enzyme.

[0010] Relevant Literature

[0011] U.S. Pat. No. 5,484,727 reports the cloning of the Human ACAT-1gene.

[0012] Farese, “Acyl CoA:cholesterol acyltransferase genes and knockoutmice,”Curr Opin Lipidol (1998 April) 9(2):119-123, provides a review ofthe current knowledge of ACAT genes.

[0013] ACAT-1 is described in Goodman, D. S., Physiol. Rev. (1965) 45:747-839; Suckling & Strange, J. Lipids Res. (1985) 26:647-671 and Changet al., Annu. Rev. Biochem. (1997) 66: 613-638; Meiner et al.,“Disruption of the acyl-CoA:cholesterol acyltransferasegene in mice:evidence suggesting multiple cholesterol esterification enzymes inmammals,” Proc Natl Acad Sci U S A (1996 Nov. 26) 93(24):14041-14046;Meiner et al., “Tissue expression studies on the mouseacyl-CoA:cholesterol acyltransferase gene (Acact): findings supportingthe existence of multiple cholesterol esterification enzymes in mice,”JLipid Res (1997 September) 38(9):1928-1933; Erickson et al.,“Acyl-coenzyme A:cholesterol acyltransferase in human liver. In vitrodetection and some characteristics of the enzyme,”Metabolism (1980,October)29(10):991-996; Tabas et al., “Acyl coenzyme A:cholesterol acyltransferase in macrophages utilizes a cellular pool of cholesteroloxidase-accessible cholesterol as substrate,”J Biol Chem (1988 Jan. 25)263(3):1266-1272; and Uelmen et al., “Tissue-specific expression andcholesterol regulation of acylcoenzyme A:cholesterol acyltransferase(ACAT) in mice. Molecular cloning of mouse ACAT cDNA, chromosomallocalization, and regulation of ACAT in vivo and in vitro,” J Biol Chem(1995 Nov. 3) 270(44):26192-26201.

[0014] The role of ACAT in various biological processes is discussed in:Field et al., Gastroenterology (1990) 99:539-551; Wilson et al., J.Lipid Res. (1994) 35:943-955; Dixon & Ginsberg, Annu. Rev. Biochem.(1993) 34:167-179; Brown & Goldstein, Annu. Rev. Biochem. (1983)52:223-261.

SUMMARY OF THE INVENTION

[0015] Nucleic acid compositions encoding novel ACAT proteins, as wellas the novel ACAT-2 proteins, (ACAT-2) are provided. Also provided aremethods of producing the subject nucleic acid and protein compositions.The subject polypeptide and nucleic acid d compositions find use in avariety of applications, including diagnostic and therapeutic agentscreening applications, as well as in treatment therapies for diseaseconditions associated with ACAT-2 activity, e.g., in the treatment ofgall stones.

DETAILED DESCRIPTION OF THE INVENTION

[0016] Nucleic acid compositions encoding novel ACAT proteins, as wellas the novel ACAT-2 proteins, (ACAT-2) are provided. Also provided aremethods of producing the subject nucleic acid and protein compositions.The subject polypeptide and nucleic acid compositions find use in avariety of applications, including diagnostic and therapeutic agentscreening applications, as well as in treatment therapies for diseaseconditions associated with ACAT-2 activity, e.g., in the treatment ofgall stones.

[0017] Before the subject invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

[0018] In this specification and the appended claims, the singular forms“a,” “an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

[0019] Nucleic Acid Compositions

[0020] Nucleic acid compositions encoding polypeptide products(hereinafter ACAT-2 and described in greater detail below), as well asfragments thereof, are provided. By nucleic acid composition is meant acomposition comprising a sequence of DNA having an open reading framethat encodes an ACAT-2 polypeptide, i.e. a gene encoding a polypeptidehaving ACAT activity, where the encoded polypeptide is not ACAT-1 asdisclosed in U.S. Pat. No. 5,484,727, the disclosure of which is hereinincorporated by reference, and is capable, under appropriate conditions,of being expressed as an ACAT-2 polypeptide. Also encompassed in thisterm are nucleic acids that are homologous, substantially similar oridentical to the nucleic acids encoding ACAT-2 polypeptides or proteins.Thus, the subject invention provides genes encoding mammalian ACAT-2,such as genes encoding human ACAT-2 and homologs thereof and mouseAcat-2 and homologs thereof.

[0021] The coding sequence of the mouse Acat-2 gene, i.e. the mouse cDNAencoding the mouse Acat-2 enzyme, has the nucleic acid sequenceidentified in U.S. patent application Ser. No. 09/328,857; thedisclosure of which is herein incorporated by reference. Acat-2 maps tomouse chromosome 15. The coding sequence of the human ACAT-2 gene, i.e.the human cDNA encoding the human ACAT-2 enzyme, has the nucleic acidsequence identified in U.S. patent application Ser. No. 09/328,857; thedisclosure of which is herein incorporated by reference. Of interest inmany embodiments is a nucleic acid (or the complement thereof) thathybridizes with either of these sequences under stringent conditions,where stringent conditions are defined below.

[0022] The source of homologous genes to those specifically listed abovemay be any mammalian species, e.g., primate species, particularly human;rodents, such as rats and mice, canines, felines, bovines, ovines,equines, yeast, nematodes, etc. Between mammalian species, e.g., humanand mouse, homologs have substantial sequence similarity, e.g. at least75% sequence identity, usually at least 90%, more usually at least 95%between nucleotide sequences. Sequence similarity is calculated based ona reference sequence, which may be a subset of a larger sequence, suchas a conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 nt long, more usually atleast about 30 nt long, and may extend to the complete sequence that isbeing compared. Algorithms for sequence analysis are known in the art,such as BLAST, described in Altschul et al. (1990), J. Mol. Biol.215:403-10, etc. Unless specified otherwise, all sequence identityvalues provided herein are determined using GCG (Genetics ComputerGroup, Wisconsin Package, Standard Settings, gap creation penalty 3.0,gap extension penalty 0.1). The sequences provided herein are essentialfor recognizing ACAT-2 related and homologous polynucleotides indatabase searches.

[0023] Nucleic acids encoding the ACAT-2 proteins and ACAT-2polypeptides of the subject invention may be cDNAs or genomic DNAs, aswell as fragments thereof. The term “ACAT-2-gene” shall be intended tomean the open reading frame encoding specific ACAT-2 proteins andpolypeptides, and ACAT-2 introns, as well as adjacent 5′ and 3′non-coding nucleotide sequences involved in the regulation ofexpression, up to about 20 kb beyond the coding region, but possiblyfurther in either direction. The gene may be introduced into anappropriate vector for extrachromosomal maintenance or for integrationinto a host genome.

[0024] The term “cDNA” as used herein is intended to include all nucleicacids that share the arrangement of sequence elements found in nativemature mRNA species, where sequence elements are exons and 3′ and 5′non-coding regions. Normally mRNA species have contiguous exons, withthe intervening introns, when present, being removed by nuclear RNAsplicing, to create a continuous open reading frame encoding an ACAT-2protein.

[0025] A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include the 3′ and 5′untranslated regions found in the mature mRNA. It may further includespecific-transcriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ or 3′ end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller, and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ or 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue and stage specific expression.

[0026] The nucleic acid compositions of the subject invention may encodeall or a part of the subject ACAT-2 proteins and polypeptides, describedin greater detail infra. Double or single stranded fragments may beobtained from the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. For the most part, DNAfragments will be at least 15 nt, usually at least 18 nt or 25 nt, andmay be at least about 50 nt. Of interest in certain embodiments arefragments which encode the N terminal portion of the encoded ACAT-2protein, as described in greater detail infra, where the N-terminalportion may be at least about the N-terminal 25, 50, 75, 80, 85, 90 or95 residues. Of interest in other embodiments are fragments which encodethe C-terminal portion of the ACAT-2 protein, where the C-terminalportion may be at least about the C-terminal 100, 200, 300, 400 or 410residues. Also of interest are nucleic acids in which the above N and Cterminal encoding fragments flank an additional nucleic acid sequence,where this additional nucleic acid sequence may be anywhere from 10 to200, usually from about 50 to 150 and more usually from about 50 to 100nucleotides in length.

[0027] The ACAT-2 genes of the subject invention are isolated andobtained in substantial purity, generally as other than an intactchromosome. Usually, the DNA will be obtained substantially free ofother nucleic acid sequences that do not include an ACAT-2 sequence orfragment thereof, generally being at least about 50%, usually at leastabout 90% pure and are typically “recombinant”, i.e. flanked by one ormore nucleotides with which it is not normally associated on a naturallyoccurring chromosome.

[0028] In addition to the plurality of uses described in greater detailin following sections, the subject nucleic acid compositions find use inthe preparation of all or a portion of the ACAT-2 polypeptides, asdescribed below.

Polypeptide Compositions

[0029] Also provided by the subject invention are polypeptides havingACAT-2 activity, i.e. capable of catalyzing the esterification ofcholesterol, as well as okysterols, with fatty acyl CoA substrates. Inaddition to being capable of catalyzing the esterification ofcholesterol with a fatty acyl CoA substrates, the subject proteins areincapable of esterifying, at least to any substantial extent, thefollowing substrates: ethanol, retinol, tocopherol, β-sitoserol,lanosterol, vitamins D1 and D2, or diacylglycerol. With respect to fattyacyl CoA substrates, the ACAT-2 polypeptides exhibit the followingpreference: palmitoyl≧linoleoyl≧oleoyl≧arachindonyl. With ACAT-2polypeptides, linoleoyl and palmitoyl compete with oleoyl forincorporation into cholesterol esters, but arachindonyl competes lesswell. In certain in vitro assays (see those reported in the ExperimentalSection, infra), the subject ACAT-2 polypeptides exhibit higher activitywith olcoyl than with palmitoyl.

[0030] The term polyeptide composition as used herein refers to both thefull length proteins as well as portions or fragments thereof. Alsoincluded in this term are variations of the naturally occurringproteins, where such variations are homologous or substantially similarto the naturally occurring protein, as described in greater detailbelow, be the naturally occurring protein the human protein, mouseprotein, or protein from some other species which naturally expresses anACAT-2 enzyme, usually a mammalian species. In the following descriptionof the subject invention, the term ACAT-2 is used to refer not only tothe human form of the enzyme, but also to homologs thereof expressed innon-human species, e.g. murine, rat and other mammalian species.

[0031] The subject ACAT-2 proteins are, in their natural environment,trans-membrane proteins. The subject proteins are characterized by thepresence of at least one potential tyrosine phosphorylation site presentin the motif MK-X-H/Y-SF, and multiple hydrophobic domains, including 5to 10, usually 6 to 9 hydrophobic domains capable of serving astrans-membrane regions. The proteins range in length from about 400 to650, usually from about 475 to 525 and more usually from about 485 to500 amino acid residues, and the projected molecular weight of thesubject proteins based solely on the number of amino acid residues inthe protein ranges from about 50 to 80, usually from about 55 to 75 andmore usually from about 60 to 65 kDa, where the actual molecular weightmay vary depending on the amount of glycolsylation, if any, of theprotein and the apparent molecular weight may be considerably less (40to 50 kDa) due to SDS binding on gels.

[0032] The amino acid sequences of the subject proteins arecharacterized by having at least some homology to a corresponding ACAT-1protein from the same species, e.g. a human ACAT-2 protein has at leastsome sequence homology with the human ACAT-1 protein, the mouse ACAT-2protein has at least some sequence homology with the mouse Acat-1protein, etc., where the sequence homology will not exceed about 80%,and usually will not exceed about 70% and more usually will not exceedabout 60%, but will be at least about 30% and more usually at leastabout 40%, as determined using GCG (Genetics Computer Group, WisconsinPackage, Standard Settings, gap creation penalty 3.0, gap extensionpenalty 0.1).

[0033] Of particular interest in many embodiments are proteins that arenon-naturally glycosylated. By non-naturally glycosylated is meant thatthe protein has a glycosylation pattern, if present, which is not thesame as the glycosylation pattern found in the corresponding naturallyoccurring protein. For example, human ACAT-2 of the subject inventionand of this particular embodiment is characterized by having aglycosylation pattern, if it is glycosylated at all, that differs fromthat of naturally occurring human ACAT-2. Thus, the non-naturallyglycosylated ACAT-2 proteins of this embodiment include non-glycosylatedACAT-2 proteins, i.e. proteins having no covalently bound glycosylgroups.

[0034] The activity of the ACAT-2 protein is inhibited by a number ofdifferent compounds, including non-specific inhibitors, such as PMSF,PHMB and progesterone, and specific inhibitors, such as PD 132301-2,CI-976 and CI-1011. ACAT-2 is more sensitive to CI-976 and CI-1011 thanACAT-1, having an IC₅₀ value that is typically less than half thatobserved with ACAT-1 in the presence of the same inhibitor.

[0035] Of particular interest in certain embodiments is the mouse ACAT-2protein, where the mouse ACAT-2 protein of the subject invention has anamino acid sequence that is substantially the same as or identical tothe sequence appearing in U.S. patent application Ser. No. 09/328,857;the disclosure of which is herein incorporated by reference. Bysubstantially the same as is meant a protein having a sequence that hasat least about 80%, usually at feast about 90% and more usually at leastabout 98% sequence identity with this sequence, as measured by GCG,supra. Of particular interest are proteins encoded by a nucleic acid (orthe complement thereof) that hybridize to the specific ACAT-2 nuclicacid sequences referenced above under stringent conditions (definedinfra).

[0036] Of particular interest in other embodiments is the human ACAT-2protein, where the human ACAT-2 protein of the subject invention has anamino acid sequence that is substantially the same as, or identical to,the sequence appearing in U.S. patent application Ser. No. 09/328,857;the disclosure of which is herein incorporated by reference. Ofparticular interest are proteins encoded by a nucleic acid (or thecomplement thereof) that hybridizes to the human nucleic acid ACAT-2sequence identified above under stringent conditions (defined infra).

[0037] In addition to the specific ACAT-2 proteins described above,homologs or proteins (or fragments thereof) from other species, i.e.other animal or plant species, are also provided, where such homologs orproteins may be from a variety of different types of species, usuallymammals, e.g. rodents, such as mice, rats; domestic animals, e.g. horse,cow, dog, cat; and humans. By homolog is meant a protein having at leastabout 35%, usually at least about 40% and more usually at least about60% amino acid sequence identity the specific ACAT-2 proteins identifiedin U.S. patent application Ser. No. 09/328,857, where sequence identityis determined using the GCG, supra.

[0038] The ACAT-2 proteins of the subject invention (e.g. human ACAT-2,mouse ACAT-2 or homologs thereof) are present in a non-naturallyoccurring environment, e.g. are separated from their naturally occurringenvironment. In certain embodiments, the subject ACAT-2 protein ispresent in a composition that is enriched for ACAT-2 as compared toACAT-2 in its naturally occurring environment. As such, purified ACAT-2is provided, where by purified is meant that ACAT-2 is present in acomposition that is substantially free of non ACAT-2 proteins, where bysubstantially free is meant that less than 90%, usually less than 60%and more usually less than 50% of the composition is made up ofnon-ACAT-2 proteins.

[0039] In certain embodiments of interest, the ACAT-2 protein is presentin a composition that is substantially free of the constituents that arepresent in its naturally occurring environment. For example, a humanACAT-2 protein comprising composition according to the subject inventionin this embodiment will be substantially, if not completely, free ofthose other biological constituents, such as proteins, carbohydrates,lipids, etc., with which it is present in its natural environment. Assuch, protein compositions of these embodiments will necessarily differfrom those that are prepared by purifying the protein from a naturallyoccurring source, where at least trace amounts of the protein'sconstituents will still be present in the composition prepared from thenaturally occurring source.

[0040] The ACAT-2 of the subject invention may also be present as anisolate, by which is meant that the ACAT-2 is substantially free of bothnon-ACAT-2 proteins and other naturally occurring biologic molecules,such as oligosaccharides, polynucleotides and fragments thereof, and thelike, where substantially free in this instance means that less than70%, usually less than 60% and more usually less than 50% of thecomposition containing the isolated ACAT-2 is a non-ACAT-2 naturallyoccurring biological molecule. In certain embodiments, the ACAT-2 ispresent in substantially pure form, where by substantially pure form ismeant at least 95%, usually at least 97% and more usually at least 99%pure.

[0041] In addition to the naturally occurring ACAT-2 proteins, ACAT-2polypeptides which vary from the naturally occurring ACAT-2 proteins arealso provided. By ACAT-2 polypeptides is meant proteins having an aminoacid sequence encoded by an open reading frame (ORF) of an ACAT-2 gene,described supra, including the full length ACAT-2 protein and fragmentsthereof, particularly biologically active fragments and/or fragmentscorresponding to functional domains; and including fusions of thesubject polypeptides to other proteins or parts thereof. Fragments ofinterest will typically be at least about 10 aa in length, usually atleast about 50 aa in length, and may be as long as 300 aa in length orlonger, but will usually not exceed about 1000 aa in length, where thefragment will have a stretch, of amino acids that is identical to anACAT-2 protein as provided in U.S. patent application Ser. No.09/328,857, or a homolog thereof; of at least about 10 aa, and usuallyat least about 15 aa, and in many embodiments at least about 50 aa inlength. In certain embodiments, N-terminal fragments of the ACAT-2protein are of interest, e.g. fragments of the N-terminal 25, 50, 75,80, 85, 90 or 95 residues. Of interest in other embodiments arefragments the C-terminal portion of the ACAT-2 protein, where theC-terminal portion may be at least about the C-terminal 100, 200, 300,400 or 410 residues. Also of interest are fusion proteins of at leastone of, or both of, the above C and N terminal fragments with anadditional polypeptide sequence. In certain embodiments, of interest isa fusion protein in which the above N and C terminal fragments flank anadditional sequence, where this additional sequence may be anywhere fromabout 3 to 75, usually from about 10 to 50 and more usually from about10 to 30 residues in length.

Preparation of ACAT-2 Polypeptides

[0042] The subject ACAT-2 proteins and polypeptides may be obtained fromnaturally occurring sources, but are preferably synthetically produced.Where obtained from naturally occurring sources, the source chosen willgenerally depend on the species from which the ACAT-2 is to be derived.

[0043] The subject ACAT-2 polypeptide compositions may be syntheticallyderived by expressing a recombinant gene encoding ACAT-2, such as thepolynucleotide compositions described above, in a suitable host. Forexpression, an expression cassette may be employed. The expressionvector will provide a transcriptional and translational initiationregion, which may be inducible or constitutive, where the coding regionis operably linked under the transcriptional control of thetranscriptional initiation region, and a transcriptional andtranslational termination region. These control regions may be native toan ACAT-2 gene, or may be derived from exogenous sources.

[0044] Expression vectors generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences encoding heterologous proteins. A selectablemarker operative in the expression host may be present. Expressionvectors may be used for the production of fusion proteins, where theexogenous fusion peptide provides additional functionality, i.e.increased protein synthesis, stability, reactivity with definedantisera, an enzyme marker, e.g. -galactosidase, etc.

[0045] Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Of particular interest is the use of sequences thatallow for the expression of functional epitopes or domains, usually atleast about 8 amino acids in length, more usually at least about 15amino acids in length, to about 25 amino acids, and up to the completeopen reading frame of the gene. After introduction of the DNA, the cellscontaining the construct may be selected by means of a selectablemarker, the cells expanded and then used for expression.

[0046] ACAT-2 proteins and polypeptides may be expressed in prokaryotesor eukaryotes in accordance with conventional ways, depending upon thepurpose for expression. For large scale production of the protein, aunicellular organism, such as E. coli, B. subtilis, S. cerevisiae,insect cells in combination with baculovirus vectors, or cells of ahigher organism such as vertebrates, particularly mammals, e.g. COS 7cells, may be used as the expression host cells. In some situations, itis desirable to express the ACAT-2 gene in eukaryotic cells, where theACAT-2 protein will benefit from native folding and post-translationalmodifications. Small peptides can also be synthesized in the laboratory.Polypeptides that are subsets of the complete ACAT-2 sequence may beused to identify and investigate parts of the protein important forfunction.

[0047] Once the source of the protein is identified and/or prepared,e.g. a transfected host expressing the protein is prepared, the proteinis then purified to produce the desired ACAT-2 comprising composition.Any convenient protein purification procedures may be employed, wheresuitable protein purification methodologies are described in Guide toProtein Purification, (Deuthser ed.) (Academic Press, 1990). Forexample, a lysate may be prepared from the original source, e.g.naturally occurring cells or tissues that express ACAT-2 or theexpression host expressing ACAT-2, and purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, and thelike.

Uses of the Subject ACAT-2 Polypeptide and Nucleic Acid Compositions

[0048] The subject polypeptide and nucleic acid compositions find use ina variety of different applications, including diagnostic andtherapeutic agent screening/discovery/preparation applications, as wellas the treatment of disease conditions associatedwith ACAT-2 activity.

General Applications

[0049] The subject nucleic acid compositions find use in a variety ofgeneral applications. General applications of interest include: (a) theidentification of ACAT-2 homologs; (b) as a source of novel promoterelements; (c) the identification of ACAT-2 expression regulatoryfactors; (d) as probes and primers in hybridization applications, e.g.PCR; (e) the identification of expression patterns in biologicalspecimens; (f) the preparation of cell or animal models for ACAT-2function; (g) the preparation of in vitro models for ACAT-2 function;etc.

[0050] Identification of ACAT-2 Homologs

[0051] Homologs of ACAT-2 are identified by any of a number of methods.A fragment of the provided cDNA may be used as a hybridization probeagainst a cDNA library from the target organism of interest, where lowstringency conditions are used. The probe may be a large fragment, orone or more short degenerate primers. Nucleic acids having sequencesimilarity are detected by hybridization under low stringencyconditions, for example, at 50° C. and 6×SSC (0.9 M sodium chloride/0.09M sodium citrate) and remain bound when subjected to washing at 55° C.in 1×SSC (0.15 M sodium chloride/0.015 M sodium citrate). Sequenceidentity may be determined by hybridization under stringent conditions,for example, at 50° C. or higher and 0.1×SSC (15 mM sodium chloride/01.5mM sodium citrate). Nucleic acids having a region of substantialidentity to the provided ACAT-2 sequences, e.g. allelic variants,genetically altered versions of the gene, etc., bind to the providedACAT-2 sequences under stringent hybridization conditions. By usingprobes, particularly labeled probes of DNA sequences, one can isolatehomologous or related genes.

[0052] Identification of Novel Promoter Elements

[0053] The sequence of the 5′ flanking region may be utilized forpromoter elements, including enhancer binding sites, that provide forregulation in tissues where A CAT-2 is expressed. The tissue specificexpression is useful for determining the pattern of expression, and forproviding promoters that mimic the native pattern of expression.Naturally occurring polymorphisms in the promoter region are useful fordetermining natural variations in expression, particularly those thatmay be associated with disease.

[0054] Identification of ACAT-2 Expression Regulatory Factors

[0055] Alternatively, mutations may be introduced into the promoterregion to determine the effect of altering expression in experimentallydefined systems. Methods for the identification of specific DNA motifsinvolved in the binding of transcriptional factors are known in the art,e.g. sequence similarity to known binding motifs, gel retardationstudies, etc. For examples, see Blackwell et al. (1995), Mol. Med.1:194-205; Mortlock et al. (1996), Genome Res. 6:327-33; and Joulin andRichard-Foy (1995), Eur. J. Biochem. 232:620-626.

[0056] The regulatory sequences may be used to identify cis actingsequences required for transcriptional or translational regulation ofACAT-2 expression, especially in different tissues or stages ofdevelopment, and to identify cis acting sequences and trans-actingfactors that regulate or mediate ACAT-2 expression. Such transcriptionor translational control regions may be operably linked to an ACAT-2gene in order to promote expression of wild type or altered ACAT-2 orother proteins of interest in cultured cells, or in embryonic, fetal oradult tissues, and for gene therapy.

[0057] Probes and Primers

[0058] Small DNA fragments are useful as primers for PCR, hybridizationscreening probes, etc. Larger DNA fragments, i.e. greater than 100 ntare useful for production of the encoded polypeptide, as described inthe previous section. For use in amplification reactions, such as PCR, apair of primers will be used. The exact composition of the primersequences is not critical to the invention, but for most applicationsthe primers will hybridize to the subject sequence under stringentconditions, as known in the art. It is preferable to choose a pair ofprimers that will generate an amplification product of at least about 50nt, preferably at least about 100 nt. Algorithms for the selection ofprimer sequences are generally known, and are available in commercialsoftware packages. Amplification primers hybridize to complementarystrands of DNA, and will prime towards each other.

[0059] Identification of Expression Patterns in Biological Specimens

[0060] The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell established in the literature. Briefly, DNA or mRNA is isolatedfrom a cell sample. The mRNA may be amplified by RT-PCR, using reversetranscriptase to form a complementary DNA strand, followed by polymerasechain reaction amplification using primers specific for the subject DNAsequences. Alternatively, the mRNA sample is separated by gelelectrophoresis, transferred to a suitable support, e.g. nitrocellulose,nylon, etc., and thenprobed with a fragment of the subject DNA as aprobe. Other techniques, such as oligonucleotide ligation assays, insitu hybridizations, and hybridization to DNA probes arrayed on a solidchip may also find use. Detection of mRNA hybridizing to the subjectsequence is indicative of ACAT-2 gene expression in the sample.

[0061] The Preparation of ACAT-2 Mutants

[0062] The sequence of an ACAT-2 gene, including flanking promoterregions and coding regions, may be mutated in various ways known in theart to generate targeted changes in promoter strength, sequence of theencoded protein, etc. The DNA sequence or protein product of such amutation will usually be substantially similar to the sequences providedherein, i.e. will differ by at least one nucleotide or amino acid,respectively, and may differ by at least two but not more than about tennucleotides or amino acids. The sequence changes may be substitutions,insertions, deletions, or a combination thereof. Deletions may furtherinclude larger changes, such as deletions of a domain or exon. Othermodifications of interest include epitope tagging, e.g. with the FLAGsystem, HA, etc. For studies of subcellular localization, fusionproteins with green fluorescent proteins (GFP) may be used.

[0063] Techniques for in vitro mutagenesis of cloned genes are known.Examples of protocols for site specific mutagenesis may be found inGustin et al: (1993), Biotechniques 14:22; Barany (1985), Gene37:111-23; Colicelli et al. (1985), Mol. Gen. Genet. 199:537-9; andPrentki et al. (1984), Gene 29:303-13. Methods for site specificmutagenesis can be found in Sambrook et al., Molecular Cloning: ALaboratory Manual, CSH Press 1989, pp. 15.3-15.108; Weiner et al.(1993), Gene 126:35-41; Sayers et al. (1992), Biotechniques 13:592-6;Jones and Winistorfer (1992), Biotechniques 12:528-30; Barton et al.(1990), Nucleic Acids Res 18:7349-55; Marotti and Tomich (1989), GeneAnal. Tech. 6:67-70; and Zhu (1989), Anal Biochem 177:120-4. Suchmutated genes may be used to study structure-function relationships ofACAT-2, or to alter properties of the protein that affect its functionor regulation.

[0064] Production of In Vivo Models of ACAT-2 Function

[0065] The subject nucleic acids can be used to generate transgenic,non-human animals or site specific gene modifications in cell lines.Transgenic animals may be made through homologous recombination, wherethe normal Acat-2 locus is altered. Alternatively, a nucleic acidconstruct is randomly integrated into the genome. Vectors for stableintegration include plasmids, retroviruses and other animal viruses,YACs, and the like.

[0066] The modified cells or animals are useful in the study of ACAT-2function and regulation. For example, a series of small deletions and/orsubstitutions may be made in the host's native ACAT-2 gene to determinethe role of different exons in cholesterol metabolism, e.g. cholesterolester synthesis, cholesterol absorption, etc. Specific constructs ofinterest include anti-sense ACAT-2, which will block ACAT-2 expression,expression of dominant negative ACAT-2 mutations, and over-expression ofACAT-2 genes. Where an ACAT-2 sequence is introduced, the introducedsequence may be either a complete or partial sequence of an ACAT-2 genenative to the host, or may be a complete or partial ACAT-2 sequence thatis exogenous to the host animal, e.g., a human ACAT-2 sequence. Adetectable marker, such as lac Z, may be introduced into the acat-2locus, where upregulation of ACAT-2 expression will result in an easilydetected change in phenotype.

[0067] One may also provide for expression of the ACAT-2 gene orvariants thereof in cells or tissues where it is not normally expressed,at levels not normally present in such cells or tissues, or at abnormaltimes of development.

[0068] DNA constructs for homologous recombination will comprise atleast a portion of the ACAT-2 gene native to the species of the hostanimal, wherein the gene has the desired genetic modification(s), andincludes regions of homology to the target locus. DNA constructs forrandom integration need not include regions of homology to mediaterecombination. Conveniently, markers for positive and negative selectionare included. Methods for generating cells having targeted genemodifications through homologous recombination are known in the art. Forvarious techniques for transfecting mammalian cells, see Keown et al.(1990), Meth. Enzymol. 185:527-537.

[0069] For embryonic stem (ES) cells, an ES cell line may be employed,or embryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

[0070] The chimeric animals are screened for the presence of themodified gene and males and females having the modification are mated toproduce homozygous progeny. If the gene alterations cause lethality atsome point in development, tissues or organs can be maintained asallogeneic or congenic grafts or transplants, or in in vitro culture.The transgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc., e.g. to determine the effectof a candidate drug on ACAT-2 activity.

[0071] Production of In Vitro Models of ACAT-2 Function

[0072] One can also use the polypeptide compositions of the subjectinvention to produce in vitro models of ACAT-2 function, e.g. theability to catalyze the esterification of cholesterol with a fatty acylCoA substrate. Such models will generally at least include the subjectACAT-2 proteins and ACAT-2 substrates, such as cholesterol and fattyacyl CoAs.

Diagnostic Applications

[0073] Also provided are methods of diagnosing disease states associatedwith ACAT-2 activity, e.g. based on observed levels of ACAT-2 or theexpression level of the ACAT-2 gene in a biological sample of interest.Samples, as used herein, include biological fluids such as semen, blood,cerebrospinal fluid, tears, saliva, lymph, dialysis fluid and the like;organ or tissue culture derived fluids; and fluids extracted fromphysiological tissues. Also included in the term are derivatives andfractions of such fluids. The cells may be dissociated, in the case ofsolid tissues, or tissue sections may be analyzed. Alternatively alysate of the cells may be prepared.

[0074] A number of methods are available for determining the expressionlevel of a gene or protein in a particular sample. Diagnosis may beperformed by a number of methods to determine the absence or presence oraltered amounts of normal or abnormal ACAT-2 in a patient sample. Forexample, detection may utilize staining of cells or histologicalsections with labeled antibodies, performed in accordance withconventional methods. Cells are permeabilized to stain cytoplasmicmolecules. The antibodies of interest are added to the cell sample, andincubated for a period of time sufficient to allow binding to theepitope, usually at least about 10 minutes. The antibody may be labeledwith radioisotopes, enzymes, fluorescers, chemiluminescers, or otherlabels for direct detection. Alternatively, a second stage antibody orreagent is used to amplify the signal. Such reagents are well known inthe art. For example, the primary antibody may be conjugated to biotin,with horseradish peroxidase-conjugated avidin added as a second stagereagent. Alternatively, the secondary antibody conjugated to aflourescent compound, e.g. fluorescein, rhodamine, Texas red, etc. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

[0075] Alternatively, one may focus on the expression of ACAT-2.Biochemical studies may be performed to determine whether a sequencepolymorphism in an ACAT-2 coding region or control regions is associatedwith disease. Disease associated polymorphisms may include deletion ortruncation of the gene, mutations that alter expression level, thataffect the activity of the protein, etc.

[0076] Changes in the promoter or enhancer sequence that may affectexpression levels of ACAT-2 can be compared to expression levels of thenormal allele by various methods known in the art. Methods fordetermining promoter or enhancer strength include quantitation of theexpressed natural protein; insertion of the variant control element intoa vector with a reporter gene such as β-galactosidase, luciferase,chloramphenicol acetyltransferase, etc. that provides for convenientquantitation; and the like.

[0077] A number of methods are available for analyzing nucleic acids forthe presence of a specific sequence, e.g. a disease associatedpolymorphism. Where large amounts of DNA are available, genomic DNA isused directly. Alternatively, the region of interest is cloned into asuitable vector and grown in sufficient quantity for analysis. Cellsthat express ACAT-2 may be used as a source of mRNA, which may beassayed directly or reverse transcribed into cDNA for analysis. Thenucleic acid may be amplified by conventional techniques, such as thepolymerase chain reaction (PCR), to provide sufficient amounts foranalysis. The use of the polymerase chain reaction is described inSaiki, et al. (1985), Science 239:487, and a review of techniques may befound in Sambrook, et al. Molecular Cloning: A Laboratory Manual, CSHPress 1989, pp.14.2-14.33. Alternatively, various methods are known inthe art that utilize oligonucleotide ligation as a means of detectingpolymorphisms, for examples see Riley et al. (1990), Nucl. Acids Res.18:2887-2890; and Delahunty et al. (1996), Am. J. Hum. Genet.58:1239-1246.

[0078] A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamnine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

[0079] The sample nucleic acid, e.g. amplified or cloned fragment, isanalyzed by one of a number of methods known in the art. The nucleicacid may be sequenced by dideoxy or other methods, and the sequence ofbases compared to a wild-type ACAT-2 sequence. Hybridization with thevariant sequence may also be used to determine its presence, by Southernblots, dot blots, etc. The hybridization pattern of a control andvariant sequence to an array of oligonucleotide probes immobilized on asolid support, as described in U.S. Pat. No. 5,445,934, or in WO95/35505, may also be used as a means of detecting the presence ofvariant sequences. Single strand conformational polymorphism (SSCP)analysis, denaturing gradient gel electrophoresis (DGGE), andheteroduplex analysis in gel matrices are used to detect conformationalchanges created by DNA sequence variation as alterations inelectrophoretic mobility. Alternatively, where a polymorphism creates ordestroys a recognition site for a restriction endonuclease, the sampleis digested with that endonuclease, and the products size fractionatedto determine whether the fragment was digested. Fractionation isperformed by gel or capillary electrophoresis, particularly acrylamideor agarose gels.

[0080] Screening for mutations in ACAT-2 may be based on the functionalor antigenic characteristics of the protein. Protein truncation assaysare useful in detecting deletions that may affect the biologicalactivity of the protein. Various immunoassays designed to detectpolymorphisms in ACAT-2 proteins may be used in screening. Where manydiverse genetic mutations lead to a particular disease phenotype,functional protein assays have proven to be effective screening tools.The activity of the encoded ACAT-2 protein may be determined bycomparison with the wild-type protein.

[0081] Diagnostic methods of the subject invention in which the level ofACAT-2 expression is of interest will typically involve comparison ofthe ACAT-2 nucleic acid abundance of a sample of interest with that of acontrol value to determine any relative differences, where thedifference may be measured qualitatively and/or quantitatively, whichdifferences are then related to the presence or absence of an abnormalACAT-2 expression pattern. A variety of different methods fordetermining the nucleic acid abundance in a sample are known to those ofskill in the art, where particular methods of interest include thosedescribed in: Pietu et al., Genome Res. (June 1996) 6: 492-503; Zhao etal., Gene (Apr. 24, 1995) 156: 207-213; Soares , Curr. Opin. Biotechnol.(October 1997) 8: 542-546; Raval, J. Pharmacol Toxicol Methods (November1994) 32: 125-127; Chalifour et al., Anal. Biochem (Feb. 1, 1994) 216:299-304; Stolz & Tuan, Mol. Biotechnol. (December 19960 6: 225-230; Honget al., Bioscience Reports (1982) 2: 907; and McGraw, Anal. Biochem.(1984) 143: 298. Also of interest are the methods disclosed in WO97/27317, the disclosure of which is herein incorporated by reference.

Screening Assays

[0082] The subject ACAT-2 polypeptides find use in various screeningassays designed to identify therapeutic agents. The screening methodswill typically be assays which provide for qualitative/quantitativemeasurements of enzyme activity in the presence of a particularcandidate therapeutic agent. For example, the assay could be an assaywhich measures the esterification activity of ACAT-2 in the presence andabsence of a candidate inhibitor agent. The screening method may be anin vitro or in vivo format, where both formats are readily developed bythose of skill in the art. Depending on the particular method, one ormore of, usually one of, the components of the screening assay may belabeled, where by labeled is meant that the components comprise adetectable moiety, e.g. a fluorescent or radioactive tag, or a member ofa signal producing system, e.g. biotin for binding to anenzyme-streptavidin conjugate in which the enzyme is capable ofconverting a substrate to a chromogenic product.

[0083] A variety of other reagents may be included in the screeningassay. These include reagents like salts, neutral proteins, e.g.albumin, detergents, etc that are used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Reagents that improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may be used.

[0084] A variety of different candidate agents may be screened by theabove methods. Candidate agents encompass numerous chemical classes,though typically they are organic molecules, preferably small organiccompounds having a molecular weight of more than 50 and less than about2,500 daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

[0085] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

ACAT-2 Nucleic Acid and Polypeptide Therapeutic Compositions

[0086] The nucleic acid compositions of the subject invention also finduse as therapeutic agents in situations where one wishes to enhanceACAT-2 activity in a host, e.g. in a mammalian host in which ACAT-2activty is sufficiently low such that a disease condition is present,etc. The ACAT-2 genes, gene fragments, or the encoded ACAT-2 protein orprotein fragments are useful in gene therapy to treat disordersassociated with ACAT-2 defects. Expression vectors may be used tointroduce the ACAT-2 gene into a cell. Such vectors generally haveconvenient restriction sites located near the promoter sequence toprovide for the insertion of nucleic acid sequences. Transcriptioncassettes may be prepared comprising a transcription initiation region,the target gene or fragment thereof, and a transcriptional terminationregion. The transcription cassettes may be introduced into a variety ofvectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and thelike, where the vectors are able to transiently or stably be maintainedin the cells, usually for a period of at least about one day, moreusually for a period of at least about several days to several weeks.

[0087] The gene or ACAT-2 protein may be introduced into tissues or hostcells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Jet injection may also be usedfor intramuscular administration, as described by Furth et al. (1992),Anal Biochem 205:365-368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992), Nature 356:152-154), where gold microprojectiles arecoated with the DNA, then bombarded into skin cells.

Methods of Modulating ACAT-2 Activity in a Host

[0088] Also provided are methods of regulating, including enhancing andinhibiting, ACAT-2 activity in a host. Where the ACAT-2 activity occursin vivo in a host, an effective amount of active agent that modulatesthe activity, e.g. reduces the activity, of ACAT-2 in vivo, isadministered to the host. In many embodiments, the active agent isACAT-2 specific, e.g., an ACAT-2 specific inhibitor which inhibits ACAT2activity but not other activities, such as ACAT-1 activity. The activeagent may be avariety of different compounds, including a naturallyoccurring or synthetic small molecule compound, an antibody, fragment orderivative thereof, an antisense composition, and the like.

[0089] Naturally occurring or synthetic small molecule compounds ofinterest include numerous chemical classes, though typically they areorganic molecules, preferably small organic compounds having a molecularweight of more than 50 and less than about 2,500 daltons. Candidateagents comprise functional groups necessary for structural interactionwith proteins, particularly hydrogen bonding, and typically include atleast an amine, carbonyl, hydroxyl or carboxyl group, preferably atleast two of the functional chemical groups. The candidate agents oftencomprise cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

[0090] Also of interest as active agents are antibodies that modulate,e.g. reduce, if not inhibit, ACAT-2 activity in the host. Suitableantibodies are obtained by immunizing a host animal with peptidescomprising all or a portion of an ACAT-2 protein, such as found in theACAT-2 polypeptide compositions of the subject invention. Suitable hostanimals include mouse, rat sheep, goat, hamster, rabbit, etc. The originof the protein immunogen may be mouse, human, rat, monkey etc. The hostanimal will generally be a different species than the immunogen, e.g.human ACAT-2 used to immunize mice, etc.

[0091] The immunogen may comprise the complete protein, or fragments andderivatives thereof. Preferred immunogens comprise all or a part ofACAT-2, where these residues contain the post-translation modifications,such as glycosylation, found on the native ACAT-2. Immunogens comprisingthe extracellular domain are produced in a variety of ways known in theart, e.g. expression of cloned genes using conventional recombinantmethods, isolation from HEC, etc.

[0092] For preparation of polyclonal antibodies, the first step isimmunization of the host animal with ACAT-2, where the ACAT-2 willpreferably be in substantially pure form, comprising less than about 1%contaminant. The immunogen may comprise complete ACAT-2, fragments orderivatives thereof. To increase the immune response of the host animal,the ACAT-2 may be combined with an adjuvant, where suitable adjuvantsinclude alum, dextran, sulfate, large polymeric anions, oil & wateremulsions, e.g. Freund's adjuvant, Freund's complete adjuvant, and thelike. The ACAT-2 may also be conjugated to synthetic carrier proteins orsynthetic antigens. A variety of hosts may be immunized to produce thepolyclonal antibodies. Such hosts include rabbits, guinea pigs, rodents,e.g. mice, rats, sheep, goats, and the like. The ACAT-2 is administeredto the host, usually intradermally, with an initial dosage followed byone or more, usually at least two, additional booster dosages. Followingimmunization, the blood from the host will be collected, followed byseparation of the serum from the blood cells. The Ig present in theresultant antiserum may be further fractionated using known methods,such as ammonium salt fractionation, DEAE chromatography, and the like.

[0093] Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. Suitable animals for production of monoclonal antibodies tothe human protein include mouse, rat, hamster, etc. To raise antibodiesagainst the mouse protein, the animal will generally be a hamster,guinea pig, rabbit, etc. The antibody may be purified from the hybridomacell supernatants or ascites fluid by conventional techniques, e.g.affmiity chromatography using ACAT-2 bound to an insoluble support,protein A sepharose, etc.

[0094] The antibody may be produced as a single chain, instead of thenormal multimeric structure. Single chain antibodies are described inJost et al. (1994) J.B.C. 269:26267-73, and others. DNA sequencesencoding the variable region of the heavy chain and the variable regionof the light chain are ligated to a spacer encoding at least about 4amino acids of small neutral amino acids, including glycine and/orserne. The protein encoded by this fusion allows assembly of afunctional variable region that retains the specificity and affinity ofthe original antibody.

[0095] For in vivo use, particularly for injection into humans, it isdesirable to decrease the antigenicity of the antibody. An immuneresponse of a recipient against the blocking agent will potentiallydecrease the period of time that the therapy is effective. Methods ofhumanizing antibodies are known in the art. The humanized antibody maybe the product of an animal having transgenic human immunoglobulinconstant region genes (see for example International Patent ApplicationsWO 90/10077 and WO 90/04036). Alternatively, the antibody of interestmay be engineered by recombinant DNA techniques to substitute the CH1,CH2, CH3, hinge domains, and/or the framework domain with thecorresponding human sequence (see WO 92/02190).

[0096] The use of Ig cDNA for construction of chimeric immunoglobulingenes is known in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987)J. Immunol. 139:3521). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest. The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91-3242. Human C regiongenes are readily available from known clones. The choice of isotypewill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity.Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human lightchain constant regions, kappa or lambda, may be used. The chimeric,humanized antibody is then expressed by conventional methods.

[0097] Antibody fragments, such as Fv, F(ab′)₂ and Fab may be preparedby cleavage of the intact protein, e.g. by protease or chemicalcleavage. Alternatively, a truncated gene is designed. For example, achimeric gene encoding a portion of the F(ab′)₂ fragment would includeDNA sequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

[0098] Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

[0099] Expression vectors include plasmids, retroviruses, YACs, EBVderived episomes, and the like. A convenient vector is one that encodesa functionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcomavirus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murineleukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Igpromoters, etc.

[0100] In yet other embodiments of the invention, the active agent is anagent that modulates, and generally decreases or down regulates, theexpression of ACAT-2 in the host. Antisense molecules can be used todown-regulate expression of ACAT-2 in cells. The anti-sense reagent maybe antisense oligonucleotides (ODN), particularly synthetic ODN havingchemical modifications from native nucleic acids, or nucleic acidconstructs that express such anti-sense molecules as RNA. The antisensesequence is complementary to the mRNA of the targeted gene, and inhibitsexpression of the targeted gene products. Antisense molecules inhibitgene expression through various mechanisms, e.g. by reducing the amountof mRNA available for translation, through activation of RNAse H, orsteric hindrance. One or a combination of antisense molecules may beadministered, where a combination may comprise multiple differentsequences.

[0101] Antisense molecules may be produced by expression of all or apart of the target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996), Nature Biotechnol. 14:840-844).

[0102] A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

[0103] Antisense oligonucleotides may be chemically synthesized bymethods known in the art (see Wagner et al. (1993), supra, and Milliganet al., supra.) Preferred oligonucleotides are chemically modified fromthe native phosphodiester structure, in order to increase theirintracellular stability and binding afnnity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases.

[0104] Among useful changes in the backbone chemistry arephosphorothioates; phosphorodithioates, where both of the non-bridgingoxygens are substituted with sulfur; phosphoroamidites; alkylphosphotriesters and boranophosphates. Achiral phosphate derivativesinclude 3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

[0105] As an alternative to anti-sense inhibitors, catalytic nucleicacid compounds, e.g. ribozymes, anti-sense conjugates, etc. may be usedto inhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 9523225, and Beigelmanet al. (1995), Nucl. Acids Res. 23:4434-42). Examples ofoligonucleotides with catalytic activity are described in WO 9506764.Conjugates of anti-sense ODN with a metal complex, e.g.terpyridylCu(II), capable of mediating mRNA hydrolysis are described inBashkin et al. (1995), Appl. Biochem. Biotechnol. 54:43-56.

[0106] As mentioned above, of particular interest in many embodimentsare agents that selectively modulate ACAT-2 activity, i.e. agents thatmodulate the activity of ACAT-2 more than the activity of ACAT-1. Inmany embodiments, of interest are agents that modulate ACAT-2 activitywith substantially no effect, including no effect, on ACAT-1 activity.Of particularly interest in many embodiments are agents that modulate byat least reducing, if not substantially inhibiting, ACAT-2 activity.

[0107] As mentioned above, an effective amount of the active agent isadministered to the host, where “effective amount” means a dosagesufficient to produce a desired result, where the desired result in thedesired modulation, e.g. enhancement, reduction, of ACAT-2 activity.

[0108] In the subject methods, the active agent(s) may be administeredto the host using any convenient means capable of resulting in thedesired inhibition of ACAT-2 activity. Thus, the agent can beincorporated into a variety of formulations for therapeuticadministration. More particularly, the agents of the present inventioncan be formulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants and aerosols.

[0109] As such, administration of the agents can be achieved in variousways, including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intracheal, etc., administration.

[0110] In pharmaceutical dosage forms, the agents may be administered inthe form of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

[0111] For oral preparations, the agents can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

[0112] The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

[0113] The agents can be utilized in aerosol formulation to beadministered via inhalation. The compounds of the present invention canbe formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

[0114] Furthermore, the agents can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. The compounds of the present invention can be administeredrectally via a suppository. The suppository can include vehicles such ascocoa butter, carbowaxes and polyethylene glycols, which melt at bodytemperature, yet are solidified at room temperature.

[0115] Unit dosage forms for oral or rectal administration such assyrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing one ormore inhibitors. Similarly, unit dosage forms for injection orintravenous administration may comprise the inhibitor(s) in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

[0116] The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

[0117] The pharmaceutically acceptable excipients, such as vehicles,adjuvants, carriers or diluents, are readily available to the public.Moreover, pharmaceutically acceptable auxiliary substances, such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like, are readily available to the public.

[0118] Where the agent is a polypeptide, polynucleotide, analog ormimetic thereof, e.g. antisense composition, it may be introduced intotissues or host cells by any number of routes, including viralinfection, microinjection, or fusion of vesicles. Jet injection may alsobe used for intramuscular administration, as described by Furth et al.(1992), Anal Biochem 205:365-368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992), Nature 356:152-154), where gold microprojectiles arecoated with the ACAT-2 DNA, then bombarded into skin cells.

[0119] Those of skill in the art will readily appreciate that doselevels can vary as a function of the specific compound, the severity ofthe symptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

[0120] The subject methods find use in the treatment of a variety ofdifferent disease conditions involving ACAT-2 activity, including bothinsufficient or. hypo-ACAT-2 activity and hyper-ACAT-2 activity. Ofparticular interest is the use of the subject invention to treatpatients suffering from disease conditions associated with hyper-ACAT-2activity, such as disease conditions associated with the presence ofelevated cholesterol ester levels, e.g. hypercholesterolemia orhyperlipidemia, including hypertriglyceridemia. In other words, ofinterest is the use of the subject invention to treat patientssuffereing from a disease condition where it is desirable to reduceACAT-2 activity. In many embodiments, the disease conditions will bethose in which it is desired to selectively inhibit ACAT-2 activity andnot ACAT-1 activity. Other disease conditions include atherosclerosis.The subject compositions may also be used to modulate the immuneresponse in a host, e.g. the immune response of macrophages, whereconditions in which such modulation is desirable include immune relateddiseases, e.g. rheumatoid arthritis, multiple sclerosis, as well asinfectious diseases, e.g. toxoplasmosis.

[0121] Of particular interest in many embodiments is the use of thesubject methods to at least reduce, if not substantially or completelyeliminate ACAT activity in the liver and/or small intestine. In theseembodiments, the subject methods are employed to at least reduce, if notsubstantially or completely eliminate, diet induced hypercholesterolemiaand/or gall stone formation in the host being treated. In addition, thesubject methods may be employed to reduce or lower plasma cholesterollevels. In these methods, of particular interest is the use of ACAT-2selective inhibitors.

[0122] By treatment is meant at least an amelioration of the symptomsassociated with the pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as hypercholesterolemia, etc.As such, treatment also includes situations where the pathologicalcondition, or at least symptoms associated therewith, are completelyinhibited, e.g. prevented from happening, or stopped, e.g. terminated,such that the host no longer suffers from the pathological condition, orat least the symptoms that characterize the pathological condition.

[0123] A variety of hosts are treatable according to the subjectmethods. Generally such hosts are “mammals” or “mammalian,” where theseterms are used broadly to describe organisms which are within the classmammalia, including the orders carnivore (e.g., dogs and cats), rodentia(e.g., mice, guinea pigs, and rats), and primates (e.g. humans,chimpanzees, and monkeys). In many embodiments, the hosts will behumans.

[0124] Kits with unit doses of the active agent, usually in oral orinjectable doses, are provided. In such kits, in addition to thecontainers containing the unit doses will be an informational packageinsert describing the use and attendant benefits of the drugs intreating pathological condition of interest. Preferred compounds andunit doses are those described herein above.

[0125] The following examples are offered primarily for purposes ofillustration. It will be readily apparent to those skilled in the artthat the formulations, dosages, methods of administration, and otherparameters of this invention may be further modified or substituted invarious ways without departing from the spirit and scope of theinvention.

Experimental

[0126] I. Mouse and Human ACAT2 was cloned and characterized asdescribed in U.S. patent application Ser. No. 09/328,857; the disclosureof which is herein incorporated by reference.

[0127] II. Further Characterization

[0128] As demonstrated above, ACAT2 expression is restricted to thesmall intestine and liver. See also Anderson, et al., J. Biol. Chem.273, 26747-26754 (1998); Cases, et al, J. Biol. Chem. 273, 26755-26764(1998); and P. Oelkers et al., J. Biol. Chem. 273, 26765-26771 (1998).Given this expression pattern and the ACAT1^(−/−) phenotype, wehypothesized that ACAT2 is the major ACAT in mouse small intestine andliver, where it plays a regulatory role in intestinal cholesterolabsorption and in the synthesis and secretion of apolipoprotein B(apoB)-containing lipoproteins. To test this hypothesis, we generatedACAT2-deficient (ACAT2^(−/−)) mice using a vector that replaced theC-terminal coding sequences of ACAT2 with neo.

[0129] Briefly, a P1 bacteriophage clone containing mouse ACAT2 wasobtained (Research Genetics), and a ˜16-kb SpeI fragment was subclonedinto pBSSKII. A sequence replacement vector was constructed in pNTKloxP[(a modified version of pNTK (see R. Mortensen, in Current Protocols inMolecular Biology F. M. Ausubel, et al., Ed.,§Eds., (John Wiley & Sons,New York, 1999), vol. 2, pp. 9.16.1-9.6.11) that was provided by Yao-WuZheng, University of California, San Francisco] by amplifying andsubcloning a 1.6-kb upstream short-arm fragment containing 3′ codingsequences (primers: sense, 5′-cgcggatccGGCTCTGCTGCTCTCCATCTTGCA-3′ (SEQID NO:01) and antisense 5′-cgcggatccgaacaTCCTGTCTCCAAACCGCAG-3′ (SEQ IDNO:02), where lower case letters indicate bases that add EcoRI and BamHIrestriction sites for cloning) and a 7-kb downstream long-arm SalIfragment containing the ACAT2 stop codon and polyadenylation signal.This vector was used to generate targeted embryonic stem cells and mice(V. L. Meiner, et al., Proc. Natl. Acad. Sci. USA 93, 14041-14046(1996)). The gene disruption was confirmed by Southern analysis ofgenomic DNA digested with SpeI and a probe (primers: sense,5′-CTGGCTGCCCACGCTGTGGTGCTC-3′ (SEQ ID NO:03) and antisense,5′-GACACAAAAGATCCCAGGCACAG3′ (SEQ ID NO:04)) located upstream of thevector sequences. Subsequent genotyping in mice was done by PCR usingprimers A (5′-GTGTGCATCTGTCTCGATATGATG-3′ (SEQ ID NO:05)), B(5′-GTCATGGACCACGACGTTCCAGGTG-3 (SEQ ID NO:06)′) and C (5′-TACCGGTGGATGTGGAATGTGTGCG-3′(SEQ ID NO:07)). PCR conditions were 35 cycles of 94° C.for 1 min, 58° C. for 1 min, and 72° C. for 2 min. A and B amplify an820-bp fragment from the wild-type allele, and A and C amplify a 500-bpfragment from the targeted allele. Reverse transcriptase-PCR todemonstrate the absence of the C-terminal ACAT2 mRNA was performed usingcDNA prepared from mouse liver and small intestine and primers (sense,(5′-GGCTGTACAGCTATGTGTATCAAG-3′ (SEQ ID NO:08), and antisense,5′-TTAGGGATGGCAGGACCAAGA-3′(SEQ ID NO:09)) specific for the deletedcoding sequences. RT-PCR of glyceraldehyde-3-phosphate dehydrogenase(G3PDH) was performed as a control to demonstrate cDNA integrity (R. V.Farese, Jr., et al., J. Lipid Res. 37,347-360 (1996)). ACAT2^(−/−) micewere viable and healthy, and offspring from heterozygous intercrosseswere found in a Mendelian distribution. The ACAT2 gene disruption wasconfirmed by Southern analysis, and the absence of intact ACAT2 mRNAexpression was demonstrated by RT-PCR of cDNA from mouse small intestineand liver.

[0130] Mice studied were of a mixed (50% C57BL/6J and 50% 129/SvJae)genetic background and were between 3-5 months of age. Mice were housedin a pathogen-free barrier facility (12-h light/12-h dark cycle) and fedeither rodent chow (Picolab 20, Purina, Saint Louis, Mo.) or a synthetichigh-fat, high-cholesterol (HF/HC) diet containing 7.5% (wt/wt) cocoabutter, 1.25% (wt/wt) cholesterol, and 0.5% (wt/wt) sodium cholate(Atherogenic Diet, ICN Biomedicals, Aurora, Ohio). For analysis ofplasma, blood was obtained from the retroorbital plexus after mice werefasted for 4 h.

[0131] Data are presented as mean±SD. For parametric data, means werecompared using t test, or analysis of variance followed by the Tukeytest. For nonparametric. data, the Mann-Whitney ranked sum test orKruskal-Wallis test followed by the Tukey test was used.

[0132] The rate of incorporation of [¹⁴C]oleoyl-CoA (Amersham) intocholesterol esters was assayed by the method of Erickson et al, J. LipidRes. 21, 930-941 (1980), with modifications as described Cases, et al.,J. Biol. Chem. 273, 26755-26764 (1998). Reactions were performed at 37°C. for 5 min with 100 μg of microsomal protein and 25 μM oleoyl-CoA(specific activity=18 μCi/μmol). Exogenous cholesterol (20 nmol) wasadded to the microsomes as phosphatidylcholine:cholesterol (4:1 molarratio) liposomes to measure apparent V_(max) activities. ACAT activityin membranes was reduced by 92% in ACAT2^(−/−) small intestine (5.1±2.5vs. 61.9±58.7 pmol cholesterol ester formed/mg prot/min for wild-typemice, P<0.05) and by 99% in ACAT2^(−/−) liver (8.8±5.2 vs. 575.3±208.9pmol cholesterol ester formed/mg prot/min for wild-type mice, P<0.01)(FIG. 1C). ACAT activity in adrenal glands, which express mainly ACAT1,was similar in wild-type and ACAT2^(−/−) mice. There was no apparentupregulation of ACAT1 activity in the small intestine or liver tocompensate for ACAT2 deficiency. These results establish that ACAT2 isthe major cholesterol esterification enzyme in mouse small intestine andliver.

[0133] Colorimetric assays were used to measure plasma lipids includingtotal cholesterol (Spectrum kit, Abbott Diagnostics, Abbott Park, Ill.),free cholesterol (WAKO Chemicals, Neuss, Germany), triglycerides(Triglycerides/GB kit, Boehlinger Mannhein, Indianapolis, Ind.), andphospholipids (Phospholipids B kit, WAKO Chemicals, Neuss, Germany).Analysis of lipids in lipoprotein fractions was performed afterseparating pooled mouse plasma samples (n=4) by fast-performance liquidchromatography (FPLC) using a Pharmacia Superose-6 column (Uppsala,Sweden) (Horie et al., J. Biol. Chem. 267, 1962-1968 (1992). Fractionswere assayed for total cholesterol, free cholesterol, phospholipids, andtriglycerides (Triglycerides, Boehringer Mannhein, Indianapolis, Ind.,USA), and cholesterol esters were calculated by subtracting values offree cholesterol from total cholesterol. For tissue lipid measurements,mice were killed by cervical dislocation and exsanguinated. Lipids wereextracted from tissues, and cholesterol ester and free cholesterolcontents were measured by gas-liquid chromatography (Schwarz, et al., J.Lipid Res. 39, 1833-1843 (1998)). Tissue triglycerides were measuredwith a kit (Triglycerides 320A, Sigma, St. Louis, Mo.) as described inJensen, et al., Am. J. Physiol. 273, R683-R689 (1997).

[0134] Plasma total cholesterol levels in chow-fed wild-type andACAT2^(−/−) mice were similar. When wild-type mice were fed a HF/HCdiet, plasma cholesterol levels increased more than two-fold, whereasplasma triglyceride levels fell (from 32±7 to 18±7, P<0.05), presumablyreflecting the replacement of triglycerides in the core ofapoB-containing lipoproteins with cholesterol esters. In contrast,ACAT2^(−/−) mice fed the HF/HC diet did not develop hypercholesterolemia(70±5 vs. 203±12 mg/dl for wild-type mice, P<0.01). In addition, theirplasma triglyceride levels on the HF/HC diet (47±11 mg/dl) were similarto those in chow-fed mice and were more than twice those of wild-typemice fed this diet (P<0.01). Heterozygous ACAT2-deficient (ACAT2^(+/−))mice fed the HF/HC diet had plasma total cholesterol levels that wereintermediate between wild-type and ACAT2^(−/−) mice (271±67 vs. 179±42vs. 93±8 mg/dl for +/+, +/−, and −/−, respectively, in a separateanalysis, P<0.05).

[0135] Analysis of the lipid composition of lipoprotein subclassesshowed that the increase in plasma total cholesterol in wild-type micefed the HF/HC diet was primarily due to increased cholesterol esters intheir apoB-containing lipoproteins [very low density lipoproteins(VLDL), intermediate density lipoproteins (IDL), and low densitylipoproteins (LDL)]. Free cholesterol levels also increased in VLDL ofwild-type mice fed this diet. In contrast, cholesterol esters and freecholesterol were not increased in these lipoproteins in ACAT2^(−/−) micefed the HF/HC diet. However, on both chow and HF/HC diets, ACAT2^(−/−)mice had more triglycerides in VLDL, IDL, and LDL than wild-type mice.Other findings included higher free cholesterol levels in highdensity-lipoproteins (HDL) of ACAT2−/− mice fed either diet, and higherphospholipid levels in VLDL and IDL of wild-type mice fed the. HF/HCdiet.

[0136] For immunoblotting, samples of pooled mouse plasma (n=4) werefractionated by FPLC using a Pharmacia Superose-6 column (Uppsala,Sweden). Fractions containing VLDL, IDL, and LDL were pooled, separatedby 4% SDS-PAGE, transferred to nitrocellulose, incubated with apolyclonal antibody that recognizes the amino terminus of apolipoprotein(McCorrnick, et al., J. Biol. Chem. 271, 11963-11970 (1996)) (a giftfrom Steven Young, Gladstone Institute of Cardiovascular Disease), andbinding of the antibodies was detected by enhanced chemiluminescence(Amersham).

[0137] Plasma levels of apoB, the primary structural apolipoprotein inVLDL, IDL, and LDL, were similar in lipoprotein fractions of chow-fedwild-type and ACAT2^(−/−) mice (FIG. 2C); apoB100 levels were slightlygreater than apoB48 levels, and both forms of apoB were highest in theLDL. When wild-type mice were fed the HF/HC diet, plasma apoB100 andapoB48 levels increased, and the ratio shifted towards more apoB48. Incontrast, plasma apoB levels in ACAT2^(−/−) mice fed the HF/HC diet weresimilar to those of chow-fed mice.

[0138] Because of the dramatic differences in the plasma lipoproteinresponse to HF/HC feeding, we examined the ultrastructuralcharacteristics of the plasma lipoproteins (d<1.063 gm/ml, whichincludes VLDL, IDL, and LDL) from wild-type and ACAT2^(−/−) mice(Non-HDL lipoproteins (d<1.063) were isolated from pooled mouse plasma(n=4) by ultracentrifugation, and samples were examined by electronmicroscopy after negative staining (Hamilton, Jr. et al., J. Lipid Res.21, 981-992 (1980))). In wild-type mice fed the HF/HC diet, thelipoproteins were characterized by large numbers of particles of typicalVLDL diameters (˜30-80 nm). Many particles exhibited a flattenedsurface, characteristic of cholesterol-ester rich lipoproteins. Incontrast, the majority of lipoproteins in ACAT2^(−/−) mice fed the HF/HCdiet were smaller (˜16-18 nm) and exhibited an electron-lucent core,characteristic of triglyceride-rich lipoproteins. Particularly strikingwas the virtual absence of VLDL-sized particles (>28 nm) in HF/HC-fedACAT2^(−/−) plasma. In wild-type and ACAT2^(−/−) mice fed a chow diet,the morphology and diameters of the lipoproteins were similar.

[0139] The HF/HC diet contains cholic acid and promotes cholelithiasisin susceptible strains of mice (DW G751). When wild-type male mice werefed this diet for 3-6 weeks, 7 of 7 developed gallstones. In contrast,none of the ACAT2^(−/−) mice (9 of 9) developed gallstones. When theHF/HC diet was fed for more than 3 months, nearly all wild-type micedeveloped gallstones, whereas gallstones were rare in ACAT2^(−/−) mice(Table 1). Gallstone formation was intermediate in ACAT2^(+/−) mice.Table 1. Resistance to gallstone formation in ACAT2^(−/−) miceSemi-quantitative analysis of gallstone formation in wild type andACAT2^(−/−) mice fed a lithogenic diet for more than 3 months. Gallstoneformation was visually scored and assigned values of high (full of largestones), medium (˜half-full), low (few stones), or none. GallstonesACAT2 Genotype High Medium Low None +/+ 8 2 3 1 +/− 0 9 1 0 −/− 0 0 3 11

[0140] We hypothesized that the protection from diet-inducedhypercholesterolemia and gallstone formation in ACAT2^(−/−) miceresulted from a block in their ability to absorb dietary cholesterol. Inchow-fed mice, cholesterol absorption, as measured by a radiolabeledtracer (Cholesterol absorption was measured by a fecal isotope ratiomethod using [4-¹⁴C]cholesterol (Amersham) and [5,6-³H]sitostanol(American Radiolabeled Chemicals, Inc., St. Louis, Mo.) as described inSchwarz, et al., J. Lipid Res. 39, 1833-1843 (1998) and Turley, et al.,Gastroenterology 107, 444-452 (1994) Non-fasted mice were dosed with[¹⁴C]cholesterol and [³H]sitostanol in medium chain-lengthtriacylglycerol oil. Feces were collected for 24 h after dosing, and theratio of ¹⁴C to ³H in aliquots of samples was used to calculate thepercent cholesterol absorption. For chow-fed mice, 0.67 μCi of[¹⁴C]cholesterol and 1.67 μCi of [³H]sitostanol were used; for mice fedthe HF/HC diet, the amounts of tracers were increased ten-fold.), wassimilar in wild-type and ACAT2^(−/−) mice. In mice fed the HF/HC diet,however, cholesterol absorption was seven-fold lower in ACAT2^(−/−) micethan in wild-type mice (10.3±3.9% vs. 1.4±0.5%, P<0.01)(On this diet,the absorption of the tracer was reduced in both groups of mice ascompared with chow-fed mice. This occurred due to dilution of the tracerby the large pool of unlabeled cholesterol supplied by the HF/HC diet.The mass of cholesterol absorbed normally increases with cholesterolfeeding, despite the decrease in tracer absorption (ES10194)).

[0141] We hypothesized that the capacity to increase dietary cholesteroluptake was reduced in ACAT2^(−/−) mice, most likely due to a reducedability to synthesize cholesterol esters for intestinal lipoproteinsynthesis and secretion. We reasoned that a difference in cholesterolabsorption capacity would be reflected in hepatic cholesterolaccumulation in mice fed the HF/HC diet. When fed this diet, wild-typemice developed fatty livers (Tissues were fixed by perfusion in 4%paraformaldehyde in phosphate buffered saline (pH 7.3). Tissues wereremoved and immersed in 2% osmium tetroxide in 0.1M sodium phosphatebuffer, pH 7.4, to stain neutral lipids. Tissues were then dehydrated inan ethanol series, transitioned into propylene oxide and embedded inEpon resin. Sections (1 μm) were cut with a glass knife and stained inwarm toluidine blue.) due to a large accumulation of cholesterol esters,whereas ACAT2^(−/−) mice had normal appearing livers with a virtualabsence of cholesterol esters in their livers. Free cholesterol levelswere also not increased in livers of ACAT2^(−/−) mice. Hepatictriglyceride concentrations were similar in wild-type and ACAT2^(−/−)mice fed the chow diet [15.4±5.5 and 11.9±3.4 μg/mg tissue for wild-typeand ACAT2^(−/−) mice (n=3 each)], but were lower in ACAT2^(−/−) mice fedthe HF/HC diet [7.9±0.5 vs. 10.9±0.9 μg/mg tissue for wild-type mice(n=3 each), P<0.01].

[0142] To further test the hypothesis that a reduced capacity forcholesterol absorption in ACAT2^(−/−) mice was effectively “shielding”the liver from cholesterol provided by the HF/HC diet, we examined theexpression of several key genes involved in hepatic cholesterolmetabolism. Mice in the fed state (three hours after the onset of thedark cycle) were killed by cervical dislocation, and liver samples wereremoved. Total RNA was extracted from tissue using Trizol (LifeTechnologies) and samples were pooled (n=3) for analysis. Northernanalysis was performed with total RNA samples (15 μg) that wereseparated by electrophoresis in 1% agarose-formaldehyde gels,transferred to nylon membranes, and hybridized with ³²P-labeled probesfor 3-hydroxy-3-methyl glutaryl coenzymeA reductase (HMGR) [a gift fromJay Horton, University of Texas-Southwestern Medical Center (UTSWMC)],low density lipoprotein receptor (LDLR) (a gift from Joachim Herz,UTSWMC), and cholesterol 7α-hydroxylase (Cyp7A) (a gift from DavidRussell,UTSWMC). Membranes were re-probed for glyceraldehyde-3-phosphatedehydrogenase (G3PDH) to normalize for differences in loading, andsignal quantification was performed using a phosphoimager (BioRadMolecular Imager FX, Hercules, Calif.) and quantitation software (BioRadQuantity One). On the chow diet, the expression levels of HMG CoAreductase and LDL receptor were similar in wild-type and ACAT2^(−/−)mice. On the HF/HC diet, the expression of these genes was reduced inwild-type mice, reflecting the accumulation of cholesterol in the liver.In contrast, the expression of these genes in ACAT2^(−/−) mice fed theHF/HC diet was similar to levels in chow-fed mice, consistent with thelack of cholesterol accumulation in ACAT2^(−/−) livers. Expressionlevels of cholesterol 7α-hydroxylase, a key enzyme involved in bile acidsynthesis, were similar in wild-type and ACAT2^(−/−) mice fed a chowdiet and were suppressed in both groups of mice fed the HF/HC diet (notshown).

[0143] The block in cholesterol esterification in ACAT2^(−/−)enterocytes might be expected to cause marked elevations in cellularfree cholesterol levels in response to the HF/HC diet. However, freecholesterol levels were increased by only 30% in the small intestine ofACAT2^(−/−) mice , and intestinal enterocytes appeared normal inhistologic sections (not shown). We hypothesized that upregulation ofABC1, a recently identified mediator of cellular cholesterol efflux(AB336, MB347, SR352), might function to excrete the free cholesterolback into the intestinal lumen.

[0144] Our studies show that ACAT2 plays a major role in mousecholesterol metabolism. ACAT2 deficiency caused a dramatic, near-totaldepletion of cholesterol esters from the apoB-containing lipoproteins.Instead these lipoproteins contained mostly triglycerides in theircores. This occurred when ACAT2^(−/−) mice were fed either a chow dietor a HF/HC diet. This result establishes that ACAT2 plays a crucial rolein synthesizing cholesterol esters for lipoprotein synthesis andsecretion in mice. Our findings indicate that apoB-containinglipoproteins can be synthesized despite the virtual absence ofcholesterol esters, addressing a long-standing debate about whethercholesterol ester synthesis is required for the synthesis and secretionof apoB-containing lipoproteins (JD1667). However, normal cholesterolester availability may be needed to synthesize and assemble largerparticles (i.e., >28 nm). Our results also address the hypothesis thatACAT1 is functionally linked to the synthesis of cholesterol esters forstorage in cytosolic droplets, whereas ACAT2 is functionally coupled tothe synthesis of cholesterol esters for lipoprotein secretion. Ourresults indicate that this is an oversimplification, as ACAT2 is thepredominant ACAT in mouse liver and was capable of synthesizingcholesterol esters for storage in in HF/HC-fed wild-type mice.

[0145] ACAT2^(−/−) mice did not become hypercholesterolemic whenchallenged with a diet containing high levels of fat and cholesterol,implicating ACAT2 as a major determinant of responsiveness to dietarycholesterol in mice. Of note, a quantitative trait locus (QTL) for theresponse of plasma VLDL- and LDL-cholesterol to HF/HC feeding has beenmapped to a region of mouse chromosome 15 containing the ACAT2 gene.Because the phenotype of this quantitative trait and ACAT2-deficiencyare similar, ACAT2 may be the responsible gene. Supporting thishypothesis, ACAT2^(+/−) mice that were fed the HF/HC diet had plasmatotal cholesterol levels that were intermediate between wild-type andACAT2^(−/−) mice. It should be noted, however, that the HF/HC dietemployed in these studies is formulated to maximize cholesterolabsorption. Therefore, the importance of ACAT2 in regulating the plasmacholesterol response at lower dietary cholesterol levels remains to bedetermined.

[0146] ACAT2 deficiency dramatically reduced gallstone formation inresponse to feeding the HF/HC diet, which contains cholic acid andpromotes lithogenesis in mice (DW G751). Moreover, our results indicatethat the gallstone resistance may be a quantitative trait, as gallstoneformation was intermediate in ACAT2^(+/−) mice. From QTL analyses inmice, seven regions on different chromosomes have been identified tocontain loci (Lith genes) that confer susceptibility to gallstoneformation (BK7729, FL224). Our results identify ACAT2, which does notmap to a previously identified locus, as an additional Lith gene. Theprevious QTL analyses might not have identified ACAT2 as asusceptibility locus if the mouse strains analyzed did not differsubstantially in ACAT2 expression. The decrease in gallstone formationin ACAT2^(−/−) mice contrasts with studies that have associateddecreased hepatic ACAT activity with increased gallstone formation(Smith, et. al., J. Lipid Res. 31, 1993-2000 (1990)). These studies havehypothesized that decreased hepatic ACAT activity results in increasedfree cholesterol excretion and lithogenicity in the bile. However, ourresults indicate that ACAT2^(−/−) mice were resistant to gallstones dueto the loss of ACAT activity in the intestine, which shielded the liverfrom the dietary cholesterol. ACAT2's role as a determinant ofresponsiveness to dietary cholesterol appears to result in part from itsrole in intestinal cholesterol absorption. Although ACAT2 was notrequired for cholesterol absorption when dietary cholesterol was low,ACAT2 deficiency limited the ability to increase cholesterol absorptioncapacity when dietary cholesterol was high. These results can beinterpreted in light of the previous finding that intestinal cholesterolabsorption requires intact chylomicron synthesis and secretion. Thisindicates that when dietary cholesterol is low, ACAT2^(−/−) mice canincorporate absorbed cholesterol into nascent chylomicrons as freecholesterol molecules at the surface of particles. However, when dietarycholesterol is increased, ACAT2^(−/−) enterocytes are unable tosynthesize cholesterol esters that would normally be added to the coresof nascent chylomicrons. As a result, the capacity to increase the massof cholesterol absorbed is limited. Thus, intestinal ACAT activity maynot be required for cholesterol absorption per se, but is necessary toincrease the capacity for cholesterol absorption in response to highlevels of dietary cholesterol. These results help to clarify thelong-standing debate on the role of ACAT in cholesterol absorption. Ourresults also are consistent with a number of studies in animals in whichACAT inhibitors were most effective in reducing cholesterol absorptionin cholesterol-fed animals (BD19) (Recently increased biliarycholesterol excretion was proposed as a mechanism that can reducedietary cholesterol absorption (ES10194). (Although this mechanismprobably accounts for the decrease in tracer absorption in mice fed theHF/HC diet in our study, it is unlikely to explain the lower cholesterolabsorption in ACAT2^(−/−) mice fed this diet).

[0147] Recently, we showed that hypercholesterolemic mice lacking ACAT1unexpectedly developed massive deposition of free cholesterol in skinand brain and lacked protection from atherosclerosis development(MA711). Although the latter findings occurred in the extreme case ofsevere hyperlipidemia and total ACAT1 deficency, these findings and thecurrent results indicate that specific inhibitors that target ACAT2rather than ACAT1 find use in the treatment of a variety of diseaseconditions. In sum, the above results show that mice lacking ACAT2 aredeficient in ACAT activity in the small intestine and liver and areprotected from diet-induced hypercholesterolemia and gallstoneformation. The contributing mechanisms include an inability toincorporate cholesterol esters into intestinal- and hepatic-derivedlipoproteins and a decreased capacity to absorb dietary cholesterol.These results indicate that ACAT2 plays an important role in theresponse to dietary cholesterol and that selective ACAT2 inhibition isuseful for preventing diet-induced hypercholesterolemia and gallstones,as well as reducing plasma cholesterol levels.

[0148] It is apparent from the above results and discussion thatpolynucleotides encoding novel mammalian ACAT-2 enzymes, as well as thenovel polypeptides encoded thereby, are provided. The subject inventionis important for both research and therapeutic applications. Using theACAT-2 probes of the subject invention, the role of ACAT-2 and itsregulation in a number of physiological processes can be studied invivo. The subject invention also provides for important new ways oftreating diseases associated with ACAT-2 activity.

[0149] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.

[0150] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1 9 1 33 DNA Artificial Sequence primer 1 cgcggatccg gctctgctgctctccatctt gca 33 2 33 DNA Artificial Sequence primer 2 cgcggatccgaacatcctgt ctccaaaccg cag 33 3 24 DNA Artificial Sequence primer 3ctggctgccc acgctgtggt gctc 24 4 23 DNA Artificial Sequence primer 4gacacaaaag atcccaggca cag 23 5 24 DNA Artificial Sequence primer 5gtgtgcatct gtctcgatat gatg 24 6 25 DNA Artificial Sequence primer 6gtcatggacc acgacgttcc aggtg 25 7 25 DNA Artificial Sequence primer 7taccggtgga tgtggaatgt gtgcg 25 8 24 DNA Artificial Sequence primer 8ggctgtacag ctatgtgtat caag 24 9 21 DNA Artificial Sequence primer 9ttagggatgg caggaccaag a 21

1.-20. (Canceled)
 21. An acyl-coenzyme A:cholesterol acyltransferase-2(ACAT-2) polypeptide present in other than its naturally occurringenvironment, wherein said ACAT-2 polypeptide comprises an amino acidsequence having at least 80% amino acid sequence identity to thesequence set forth in SEQ ID NO:12or SEQ ID NO:13.
 22. The ACAT-2polypeptide of claim 21, wherein said ACAT-2 polypeptide comprises anamino acid sequence having at least 90% amino acid sequence identity tothe sequence set forth in SEQ ID NO:12 or SEQ ID NO:13.
 23. The ACAT-2polypeptide of claim 21, wherein said ACAT-2 polypeptide comprises anamino acid sequence having at least 98% amino acid sequence identity tothe sequence set forth in SEQ ID NO:12 or SEQ ID NO:13.
 24. The ACAT-2polypeptide of claim 21, wherein said ACAT-2 polypeptide comprises theamino acid sequence set forth in SEQ ID NO:12.
 25. The ACAT-2polypeptide of claim 21, wherein said ACAT-2 polypeptide comprises theamino acid sequence set forth in SEQ ID NO:13.
 26. The ACAT-2polypeptide of claim 21, wherein said ACAT-2 polypeptide catalyzesesterification of a cholesterol in the presence of a fatty acyl CoAsubstrate.
 27. A composition comprising the polypeptide of claim
 21. 28.A fragment of the ACAT-2 polypeptide of claim 21, wherein said fragmentcatalyzes esterification of a cholesterol in the presence of a fattyacyl CoA substrate.
 29. An in vitro method of identifying an agent thatinhibits an enzymatic activity of an acyl-coenzyme A:cholesterolacyltransferase-2 (ACAT-2) polypeptide, the method comprising: a)contacting the ACAT-2 polypeptide of claim 21 with a candidate agent;and b) determining the effect of the agent on enzymatic activity of theACAT-2 polypeptide.
 30. The method of claim 29, wherein said determiningcomprises measuring esterification of a cholesterol in the presence of afatty acyl CoA substrate.
 31. The method of claim 30, wherein saiddetermining comprises measuring incorporation of a detectably labeledoleoyl-CoA into a cholesterol ester.